Synthesis of glp-1 peptides

Abstract

Disclosed are processes for the synthesis of GLP-1 peptides, such as liraglutide and semaglutide, and a process for purifying liraglutide.

1 . A process for preparing a GLP-1 peptide containing a lysine having a derivatized side chain, wherein the process comprises liquid or solid phase peptide synthesis or a combination thereof, wherein the process comprises a final coupling step in which at least two fragments are coupled at a terminal Gly residue, and wherein at least one of the fragments is prepared by coupling of at least two sub-fragments. 
     
     
         2 .- 36 . (canceled) 
     
     
         37 . The process according to  claim 1 , wherein the GLP-1 peptide is semaglutide consisting of the following formula: 
       
         
           
                 
                 
               
                    1   2   3   4   5   6   7   8   9   10  11  12  13  14  15  16  17  18  19 
                     
                 
                   His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala- 
                 
                     
                 
                    20         21  22  23  24  25  26  27  28  29  30  31 
                 
                   Lys(W)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH 
                 
             
                
                
                
                
                
               
            
           
         
         wherein W=N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl [SEQ ID NO: 174] 
       
     
     
         38 .- 104 . (canceled) 
     
     
         105 . A process for preparing semaglutide, wherein the process comprises conjugating a peptide selected from the group selected from one or more of Peptide 1, P1-His(P)-Aib-Glu(P)-Gly-O-P2 (SEQ ID NO:177), Peptide 3, P1-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-Tyr(P)-Leu-Glu(P)-Gly-OP2 (SEQ ID NO: 43), and Peptide 4, P1-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp-Leu-Val-Arg(P)-Gly-Arg(P)-Gly-O-P2 (SEQ ID NO:196) with at least one additional peptide fragment, wherein P1 for Peptide 1 is a protecting group, each P in Peptide 1 represents a side chain protecting group which may be the same or different, and P2 for Peptide 1 is H, a solid support, or an activated carboxylic ester of the Gly 4  residue, wherein P1 for Peptide 3 is a protecting group, each P in Peptide 3 represents a side chain protecting group which may be the same or different and P2 for Peptide 3 is H, is an activated carboxylic ester of the Gly 16  residue, or is a solid support, and wherein P1 in Peptide 4 is H or a protecting group for the N-terminal of Gln, each P of Peptide 4 is a side chain protecting group which may be the same or different, P2 in Peptide 4 is H or a solid support, and W1 in Peptide 4 is N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetic acid. 
     
     
         106 . A composition prepared by the process of  claim 105 , wherein the composition comprises semaglutide and
 (a) less than 1 wt % of the D-His isomer of semaglutide, and/or   (b) less than 1 wt % of the [+Glyl 16 ] derivative of semaglutide, and/or   (c) less than 1 wt % of the [+Gly 31 ] derivative of semaglutide, and/or   (d) less than 1 wt % of the [+Gly 4 ] derivative of semaglutide.   
     
     
         107 . The process of  claim 105 , wherein the process comprises conjugating Peptide 3 (SEQ ID NO:43) and Peptide 4 (SEQ ID NO:196) to form Peptide 2 (SEQ ID NO:176). 
     
     
         108 . The process of  claim 107 , wherein the process further comprises conjugating Peptide 2 (SEQ ID NO:176) and Peptide 1 (SEQ ID NO:177) to form semaglutide (SEQ ID NO:174). 
     
     
         109 . The process of  claim 105 , wherein the process further comprises conjugating Peptide 4 (SEQ ID NO:196) with one additional peptide fragment to form semaglutide (SEQ ID NO:174). 
     
     
         110 . The process of  claim 105 , wherein the protecting groups and resin is removed to form semaglutide. 
     
     
         111 . The process of  claim 110 , wherein semaglutide is purified. 
     
     
         112 . A process for preparing semaglutide, wherein the process comprises conjugating a peptide selected from the group selected from one or more of Peptide 1, P1-His(P)-Aib-Glu(P)-Gly-O-P2 (SEQ ID NO:177), Peptide 5, P1-Thr(P)-Phe-Thr(P)-Ser(P)-Asp(P)-Val-Ser(P)-Ser(P)-OP2 (SEQ ID NO:78), Peptide 6, P1-Tyr(P)-Leu-Glu(P)-Gly-OP2 (SEQ ID NO: 84), and Peptide 4, P1-Gln(P)-Ala-Ala-Lys(W1)-Glu(P)-Phe-Ile-Ala-Trp-Leu-Val-Arg(P)-Gly-Arg(P)-Gly-O-P2 (SEQ ID NO:196) with at least one additional peptide fragment, wherein P1 for Peptide 1 is a protecting group, each P in Peptide 1 represents a side chain protecting group which may be the same or different, and P2 for Peptide 1 is H, a solid support, or an activated carboxylic ester of the Gly 4  residue, wherein P1 for Peptides 5 and 6 are a protecting group, each P in Peptides 5 and 6 represent a side chain protecting group which may be the same or different and P2 for Peptides 5 and 6 is H, is an activated carboxylic ester of the Gly residue, or is a solid support, and wherein P1 in Peptide 4 is H or a protecting group for the N-terminal of Gln, each P of Peptide 4 is a side chain protecting group which may be the same or different, P2 in Peptide 4 is H or a solid support, and W1 in Peptide 4 is N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetic acid. 
     
     
         113 . The process of  claim 112 , wherein the process further comprises conjugating Peptide 5 (SEQ ID NO:78) and Peptide 6 (SEQ ID NO:84) to form Peptide 3 (SEQ ID NO:43). 
     
     
         114 . The process of  claim 113 , wherein the process further comprises conjugating Peptide 3 (SEQ ID NO:43) with at least one additional peptide fragment to form semaglutide (SEQ ID NO:174). 
     
     
         115 . The process of  claim 114 , wherein the process further comprises conjugating Peptide 3 (SEQ ID NO:43) with Peptide 4 (SEQ ID NO:177) and at least one additional peptide fragment to form a semaglutide. 
     
     
         116 . The process of  claim 114 , wherein the process further comprises conjugating Peptide 3 (SEQ ID NO:43) with Peptide 4 (SEQ ID NO:196) and Peptide 1 (SEQ ID NO:177) to form semaglutide. 
     
     
         117 . The process of  claim 112 , wherein the protecting groups and resin is removed to form semaglutide. 
     
     
         118 . The process of  claim 117 , wherein semaglutide is purified. 
     
     
         119 . A composition prepared by the process of  claim 112 , wherein the composition comprises semaglutide and
 (a) less than 1 wt % of the D-His isomer of semaglutide, and/or   (b) less than 1 wt % of the [+Glyl 16 ] derivative of semaglutide, and/or   (c) less than 1 wt % of the [+Gly 31 ] derivative of semaglutide, and/or   (d) less than 1 wt % of the [+Gly 4 ] derivative of semaglutide.